We propose to investigate the effect of hormones on carbohydrate metabolism in isolated rat hepatocytes. Special emphasis will be placed on the effect of glucogon and insulin on hepatocyte ion fluxes and the relationship of these hormone-induced ion fluxes to the control of gluconeogenesis. Ion flux and/or redistribution will be studied by tracer techniques and the data subjected to compartmental analysis by means of graphical techniques and by analog computer. Control points in the gluconeogenic pathway will be evaluated by metabolite analysis and crossover plots. Since evidence supports an action of glucagon and insulin on oxidative decarboxylation of pyruvate, the role of pyruvate dehydrogenase as a possible control point in gluconeogenesis will be studied. Changes in the relative amounts of the active dephosphorylated and of the inactive, phosphorylated forms will be correlated with changes in flux through gluconeogenesis and in ion fluxes. Calcium will be of particular interest since the PDH phosphatase is Ca to the 2nd power ion dependent. Phosphodiesterase can also be activated by a Ca to the 2nd power ion minus requiring mechanism and the influence of this ion on the hormonal regulation of cyclic AMP levels will also be studied. The role of cyclic nucleotides in the mediation of hormonally induced ion fluxes will also be investigated. Possible effects of hormones and ions on the flux of carbon through pyruvate kinase will also be studied. BIBLIOGRAPHIC REFERENCES: Pilkis, S.J., Claus, T.H., Johnson, R.A., and Park, C.R. (1975) Hormonal Control of Cyclic AMP and Gluconeogenesis in Isolated Hepatocytes. J. Biol. Chem. 250, 6328-6336. Claus, T.H., Pilkis, S.J., and Park, C.R. (1975) Stimulation by Glucagon of the Incorporation of U-C14-labelled Substrates into Glucose by Hepatocytes from Fed Rats. Biochem. Biophys. Acta 404, 110-123.